Structural basis of negative regulation of CRISPR-Cas7-11 by TPR-CHAT

CRISPR‒Cas7-11 is a Type III-E CRISPR-associated nuclease that functions as a potent RNA editing tool. Tetratrico-peptide repeat fused with Cas/HEF1-associated signal transducer (TPR-CHAT) acts as a regulatory protein that interacts with CRISPR RNA (crRNA)-bound Cas7-11 to form a CRISPR-guided caspase complex (Craspase). However, the precise modulation of Cas7-11’s nuclease activity by TPR-CHAT to enhance its utility requires further study. Here, we report cryo-electron microscopy (cryo-EM) structures of Desulfonema ishimotonii (Di) Cas7-11-crRNA, complexed with or without the full length or the N-terminus of TPR-CHAT. These structures unveil the molecular features of the Craspase complex. Structural analysis, combined with in vitro nuclease assay and electrophoretic mobility shift assay, reveals that DiTPR-CHAT negatively regulates the activity of DiCas7-11 by preventing target RNA from binding through the N-terminal 65 amino acids of DiTPR-CHAT (DiTPR-CHATNTD). Our work demonstrates that DiTPR-CHATNTD can function as a small unit of DiCas7-11 regulator, potentially enabling safe applications to prevent overcutting and off-target effects of the CRISPR‒Cas7-11 system.


INTRODUCTION
2][3][4] The CRISPR-Cas system has revolutionized the field of genetic engineering and has become a widely used tool in biological research. 5Its gene editing capabilities have made it the preferred method for editing genes in a wide range of organisms, including plants, animals, and even human cells. 6,7 CRISPR-Cas system consists of two main components: a Cas protein, which is the catalytic core, and CRISPR loci, which have "genetic memory" functions. 8Based on the composition of the Cas protein and properties of the effector complexes, CRISPR-Cas systems are classified into Class 1 and Class 2 systems. 9The Class 1 system, including Types I, III, and IV, is characterized by an effector complex composed of four to seven Cas protein subunits and mediates foreign nucleic acid interference through the action of multiple Cas protein complexes. 9,10In contrast, a Class 2 system, including Types II, V, and VI, carries a single multidomain effector protein. 104][15] Cas7-11 is a single effector fusion protein, comprising four Cas7 proteins and one Cas11 protein, but it lacks the signature Cas10 protein common to Type III systems. 13Studies have shown that Cas7-11 processes precursor CRISPR RNA (pre-crRNA) and cleaves target RNA at two distinct sites separated by six nucleotides. 13,14In contrast to Cas13 family proteins, Type VI effectors that also cleave RNA, Cas7-11 exhibits minimal off-target cleavage activity, high specificity, and low levels of cytotoxicity. 13,16,17Importantly, the Type III-E system encodes additional components, including a caspase-like protease named TPR-CHAT (or Csx29), along with three accessory proteins: Csx30, Csx31, and RpoE (Fig. 1a). 13,149][20][21][22][23][24] While the Cas7-11 system holds potential as an RNA editing tool, further studies are needed to precisely modulate its cleavage activity for broader applications, especially in identifying endogenous regulators of this system.
We analyzed the available regulatory elements and found that TPR-CHAT shows the potential to be used as an accessory protein for regulating Cas7-11-mediated RNA editing.In this study, we present high-resolution cryo-EM structures of crRNA bound DiCas7-11 (DiCas7-11-crRNA complex) alone or in complex with full length or the N-terminal domain of TPR-CHAT (DiCas7-11-crRNA-TPR-CHAT or DiCas7-11-crRNA-TPR-CHAT NTD ) and the crRNA bound catalytically inactivated mutant DiCas7-11 (DiCas7-11 Inactive -crRNA complex).These elucidated structures, combined with biochemical analysis, demonstrated that the NTD fragment of TPR-CHAT functions as a small regulatory unit sufficient to inhibit Cas7-11 activity.Specifically, TPR-CHAT NTD stabilizes the conformation of Cas7-11 and prevents the binding of target RNA.Our results provide implications for understanding the precise regulation of the Cas7-11 system and advancing the application of this system.

DiTPR-CHAT inhibits the cleavage activity of DiCas7-11
To uncover the mechanism by which DiTPR-CHAT inhibits DiCas7-11 cleavage, we first determined the interaction between DiTPR-CHAT and DiCas7-11 through pulldown assays.We conducted pulldown assays using N-terminal His-tagged DiCas7-11 and C-terminal Streptagged DiTPR-CHAT (Supplementary Fig. 1a, b, d, e).Strep-tagged DiTPR-CHAT pulled down purified His-tagged DiCas7-11, regardless of whether the proteins were co-expressed or expressed separately, indicating the formation of a stable complex between DiCas7-11 and DiTPR-CHAT (Supplementary Fig. 1c, f).Subsequently, we validated the inhibitory effect of DiTPR-CHAT on DiCas7-11 using in vitro nuclease assays by incubating the purified DiCas7-11-crRNA with 5'-FAM-labeled target RNA in the presence or absence of DiTPR-CHAT (Fig. 1b and Supplementary Fig. 1g).We found that DiCas7-11-crRNA specifically cleaved the target RNA into two fragments, and the presence of DiTPR-CHAT inhibited this cleavage (Fig. 1c).The inhibitory effect of DiTPR-CHAT is more prominent when preincubating DiCas7-11-crRNA with TPR-CHAT before adding the target RNA (Supplementary Fig. 1g), suggesting that DiTPR-CHAT function as a negative regulator by pre-occupying the active site of DiCas7-11.
The high-resolution structures unveil atomic details of crRNAbound DiCas7-11 complexed with full length DiTPR-CHAT or DiTPR-CHAT NTD (Fig. 2b-d).DiCas7-11 consists of four Cas7 domains (Cas7.1-Cas7.4), a Cas11 domain, and a C-terminal extension (CTE) domain (Fig. 2a-e).Cas7.4 is unique by carrying a large insertion (INS) domain that is absent in the other three Cas7 domains (Fig. 2a-d and Supplementary Fig. 6b).The Cas7 domains, arranged in a row, form a crescent-shaped filament, with the INS and CTE domain completing the structure.The Cas11 domain is positioned at the crescent's midpoint, and interacts with Cas7.2 and Cas7.3 (Fig. 2b-d and Supplementary Fig. 6c).Cas11 is linked between Cas7.1 and Cas7.2, separated by interspacing linker 1 and 2 (L1 and L2).Cas7.2, Cas7.3 and Cas7.4 are interspaced by L3 and L4.While L1 is disordered in the structures, the remaining linkers are well resolved (Fig. 2a-d and Supplementary Fig. 6a).The crRNA is resolved in all three structures, traversing the Cas7 filament from NTD to the C-terminal domain (CTD) (Fig. 2b-d).The regulatory complex DiTPR-CHAT binds to the side of the DiCas7-11 complex, with the CHAT domain interacting with Cas7.1 and Cas7.2, and the NTD of the TPR domain associated at the interface of Cas7.3, Cas7.4 and Cas11 (Fig. 2c and Supplementary Fig. 6d).The truncated TPR-CHAT containing only the NTD can independently interact with the complex at the same position.For obtaining target RNA in the complex, we also obtained the structure using inactivated DiCas7-11-crRNA complex (DiCas7-11 Inactive ) by substituting the conserved catalytic residues, Cas7.2 D429 and Cas7.3 D654 , with alanine to prevent cleavage of bound target RNA (Fig. 2e and Supplementary Figs.2c, f, i, 5).However, the resulting complex did not reveal target RNA and exhibited no marked conformational changes to other structures (Fig. 2e and Supplementary Fig. 5).
Our DiCas7-11-crRNA and DiCas7-11-crRNA-TPR-CHAT structures show a high resemblance with a root-mean-square deviation  8a-c), implicating that the binding of TPR-CHAT did not perturb the conformation of Cas7-11.This characterization is consistent with those recently reported structures from Desulfonema ishimotonii (Supplementary Fig. 8d-f) 21 and Candidatus 'Scalindua brodae' (Sb) (Supplementary Fig. 8g-i), 26 suggesting that the regulation of Cas7-11 by TPR-CHAT may be mediated by minor conformational changes.
We further performed a pulldown assay to analyze the interactions between DiCas7.4 and DiTPR-CHAT NTD .Deletion of V1317-R1336 in Cas7.4 disrupted complex formation between DiCas7-11 and DiTPR-CHAT NTD .Similarly, mutations of the interacting residues L50A, R53A, and R64A of DiTPR-CHAT NTD at the interface also disrupted the complex formation with DiCas7-11  4e and Supplementary Fig. S7b).In contrast, the mutations of the NTD residues and G49A that formed hydrogen bonds with S1352 and H1337, respectively, outside of the segment of V1317-R1336 in Cas7.4 neither affected the formation of the complex nor the inhibitory activity of DiTPR-CHAT NTD (Fig. 4e, f and Supplementary Fig. S7b).These suggest that DiTPR-CHAT NTD regulates the activity of DiCas7-11 primarily through the segment of V1317-R1336 in Cas7. 4. A recent study also demonstrated the regulatory function of truncated NTD of DmTPR-CHAT on DmCas7-11 through interactions with the insertion finger in Cas7.4 (Supplementary Fig. 11a-c). 18This similar interactive mechanism indicates the importance of TPR-CHAT NTD for Cas7-11 regulation, although the sequences and structures of DiTPR-CHAT NTD and DmTPR-CHAT NTD are poorly conserved (Supplementary Fig. 11d-g).Surprisingly, the same mutations carried out on the full length DiTPR-CHAT did not affect the formation of complexes with DiCas7-11 or the inhibitory effect of DiTPR-CHAT (Supplementary Fig. 10a-d).This suggests that the full length DiTPR-CHAT also interacts with Cas7-11 through interfaces other than that between Cas7.4 and DiTPR-CHAT NTD .Indeed, the structure of DiCas7-11-crRNA-TPR-CHAT also exhibits interactions between L2 and CHAT domains in additional to those in the NTD (Supplementary Fig. S7a), which explains the stabilized conformation of L2 observed in the structure.
DiTPR-CHAT NTD inhibits the binding of target RNA to DiCas7-11-crRNA It is reported that the RNA cleavage site of DiCas7-11 is situated around Cas7.2 and Cas7.3. 13,14Therefore, the modulation of DiTPR-CHAT NTD in Cas7.4 would more likely impact the binding but not the cleavage of target RNA.To test this, we expressed and evaluated the affinity between the labeled target RNA and the inactivated DiCas7-11 (mutated catalytic residues at D429/D654) in the presence or absence of the full length, NTD or CTD of TPR-CHAT using electrophoretic mobility shift assays (EMSA) (Supplementary Fig. 9c, d, f).We found that the affinity of the DiCas7-11-crRNA complex to the target RNA was reduced by the presence of full length (F) or NTD of DiTPR-CHAT but unaffected by the CTD (Fig. 5a and Supplementary Fig. 1h).These results demonstrated that both DiTPR-CHAT and DiTPR-CHAT NTD blocked the binding of the target RNA to DiCas7-11, indicating that binding of DiTPR-CHAT, mainly via its NTD, reduced target RNA binding and inhibited cleavage activity, probably through modulating the conformation of the DiCas7-11-crRNA complex.DiTPR-CHAT NTD was able to inhibit the activity of Cas7-11 in a concentrationdependent manner (Fig. 5b and Supplementary Fig. 9e, g), indicating that the NTD of DiTPR-CHAT is sufficient to act as an inhibitory regulator for the activity of DiCas7-11.Based on these findings, we propose a model for the regulation of DiCas7-11 by DiTPR-CHAT that DiCas7-11 in a relaxed conformation initially binds to crRNA, which then facilitates the binding of the target RNA to crRNA for cleavage (Fig. 5c).When DiTPR-CHAT binds to this Cas7-11-crRNA complex, it stabilizes DiCas7-11-crRNA, primarily through DiTPR-CHAT NTD , causing the complex to adopt a compact conformation (Fig. 5c).This conformational change inhibited the binding of target RNA, leading to the reduced nuclease activity (Fig. 5c).

DISCUSSION
The type III-E CRISPR-Cas system is a newly identified bifunctional complex with nuclease activity and protease activity that holds great potential for RNA editing applications. 9,1522][23][24][26][27][28] However, the poor understanding of the precise modulation of Cas7-11 by TPR-CHAT limits the application of the system.
In this study, we first demonstrated that DiCas7-11 possesses cleavage activity towards target RNA guided by crRNA and that DiTPR-CHAT can inhibit the cleavage activity of DiCas7-11.To gain further insights into the mechanism of DiTPR-CHAT regulation of DiCas7-11, we determined the high-resolution structures of the DiCas7-11-crRNA and DiCas7-11-crRNA-TPR-CHAT complexes (both full-length and N-terminal domain of DiTPR-CHAT) using cryo-EM.Structural analysis revealed that the binding of DiTPR-CHAT did not induce significant conformational changes in the overall configuration of DiCas7-11-crRNA complex, but it did  11a-c), which suggest that the regulatory mechanism may be conserved among different species. 18,21][35] Although the current study on the DiCas7-11 system shows little off-target effects, 13 it is important to consider the potential occurrence of off-target effects as the system becomes more widely used, which could impact its safety.Therefore, investigating inhibitors that target DiCas7-11 is necessary.DiTPR-CHAT NTD , a truncated form of DiTPR-CHAT with a smaller molecular weight but similar function, represents an ideal protein inhibitor against DiCas7-11.It holds significant potential for various applications in gene-editing technology.However, it is noteworthy that the activity of DiTPR-CHAT NTD has only been demonstrated in vitro, and whether it can exert a similar inhibitory effect in vivo, particularly in mammalian cells, requires further study.Thus, the development of TPR-CHAT NTD as a regulator to control Cas7-11 activity biologically requires additional refinement.
In summary, our findings highlight the role of DiTPR-CHAT NTD in negatively regulating the nuclease activity of DiCas7-11 by blocking the binding of target RNA.Understanding the negative regulation of DiCas7-11 by DiTPR-CHAT is crucial for deciphering the complex dynamics of bacterial immune responses and may have implications for the development of novel strategies to modulate CRISPR-Cas activity.Future research efforts will focus on adapting and optimizing this system for use in RNA editing, with the goal of developing it as a therapeutic tool and gene detection application for clinical use.
For the expression and purification of the DiCas7-11-crRNA-TPR-CHAT complex, the two prepared plasmids (pET28a-His-SUMO-TEV-DiCas7-11-TPR-CHAT-2×Strep and pACYCDuet-1-crRNA) were cotransformed into the E. coli BL21 (DE3) strain.A single colony was grown in LB medium supplemented with 50 μg ml −1 kanamycin and 25 μg ml −1 chloramphenicol at 37 °C.Protein expression was induced by 0.5 mM IPTG at 18 °C for 14-16 h until the OD 600 nm reached 0.8.Cell pellets were then collected by centrifugation and resuspended in buffer A supplemented with 1 mM PMSF.The cells were lysed and the supernatant was collected after centrifugation at 18,000 × g for 45 min at 4 °C.The supernatant containing the DiCas7-11-crRNA-TPR-CHAT complex was purified using a Ni-NTA column (GE Healthcare) in buffer A supplemented with 250 mM imidazole.The eluted protein was further purified by chromatography on a HiTrap Strep column system (GE Healthcare) equilibrated with buffer B. The protein was eluted with 10 mM D-desthiobiotin (Sigma) and then purified on a Superdex 200 Increase 10/300 column (GE Healthcare) pre-equilibrated with buffer C. The collected peak fractions were designated for cryo-EM sample preparation or stored at −80 °C until required.
For the expression and purification of the DiCas7-11 Inactive -crRNA complex, the constructed plasmid (pET28a-His-SUMO-TEV-DiCas7-11 Inactive ) was transformed into the E. coli BL21 (DE3) strain and cultivated overnight in LB medium containing 50 μg ml −1 kanamycin.A single colony was cultured in LB medium containing 50 μg ml −1 kanamycin at 37 °C.Protein expression was induced by adding 0.5 mM IPTG and maintained at 18 °C for 14-16 h until the OD600 nm reached 0.8.Cell pellets were harvested after centrifugation and resuspended in buffer A containing 1 mM PMSF.Cells were lysed and the supernatant harvested by centrifugation at 18,000 × g for 45 min at 4 °C.The supernatant was purified using a Ni-NTA column (GE Healthcare) with buffer A containing 250 mM imidazole.The eluted protein was further purified by chromatography on a HiTrap Heparin column (GE Healthcare) equilibrated with buffer B. The protein was eluted with a linear gradient of 0-2 M NaCl.The peak fractions were collected and further purified on a Superdex 200 Increase 10/300 column (GE Healthcare) equilibrated with buffer C. For cryo-EM sample preparation, the purified DiCas7-11 Inactive sample was incubated with single spacer pre-crRNA (5'-GGUUGGAAAGCCGGUUUUCUUU-GAUGUCACGGAACCUUUGUUGUCUUCGACAUGGGUAAUCCUCAU-3') at a molar ratio of 1:2 for 1 h at room temperature.

In vitro nuclease assays
In vitro nuclease assays were carried out using purified DiCas7-11-crRNA-DiTPR-CHAT complex (for fixed-dose at 233 nM) or individually purified DiCas7-11-crRNA preincubated with DiTPR-CHAT (for increasing doses) in a nuclease assay buffer (40 mM Tris-HCl, pH 7.5, 60 mM NaCl, and 6 mM MgCl 2 ) mented with 4 of an RNase inhibitor.10 nM 5-carboxyfluorescein (5'-FAM)-labeled target RNA was added to the system and incubated at 37 °C for 30 min.Reactions with different assembly orders of the components were also carried out.To terminate the reactions, 1.5 mg ml −1 proteinase K, 6 mM EDTA, and 500 mM urea were added and incubated at 50 °C for 30 min.The samples were then heated at 95 °C for 3 min and subjected to electrophoresis on a urea gel at 180 V for 35 min at room temperature.The gels were scanned using an iBright FL1500 Imaging System (Thermo Fisher Scientific).

Protein pulldown assays
To characterize the interaction between DiCas7-11 and DiTPR-CHAT in vivo, the pET28a-His-SUMO-TEV-DiCas7-11-TPR-CHAT-2×Strep plasmid was transformed into the E. coli BL21 (DE3) strain and cultured overnight in LB medium supplemented with 50 μg ml −1 kanamycin.A single colony was cultured in LB medium supplemented with 50 μg ml −1 kanamycin at 37 °C.The protein overexpression and purification protocols were performed as previously described.The complex was identified via SDS-PAGE and Coomassie Brilliant Blue staining.To identify the interaction between DiCas7-11 and DiTPR-CHAT in vitro, the codon-optimized sequence of DiTPR-CHAT (residues 1-751), carrying a 2×Strep tag at the CTD, was amplified by PCR and cloned into the pET28a vector (pET28a-DiTPR-CHAT-2×Strep).The plasmids pET28a-His-SUMO-TEV-DiCas7-11 and pET28a-DiTPR-CHAT-2×Strep were separately transformed into the E. coli BL21 (DE3) strain and cultured overnight in LB medium supplemented with 50 μg ml −1 kanamycin.A single colony from each transformation was cultured in individual LB medium containing 50 μg ml −1 kanamycin at 37 °C.The expression of DiCas7-11 and DiTPR-CHAT was induced with IPTG, followed by their purification using a Ni-NTA column (GE Healthcare) and a HiTrap Strep column (GE Healthcare), respectively.Imidazole was removed using a desalting column (Bio-Rad).His-tagged DiCas7-11 and Strep-tagged DiTPR-CHAT were co-incubated at 4 °C for 1 h at a 1:1 molar ratio.The mixtures were then re-purified using a Ni-NTA column (GE Healthcare) and HiTrap Strep column (GE Healthcare).The eluted protein was further purified on a Superdex 200 Increase 10/300 column (GE Healthcare), and the peak fraction was collected for SDS-PAGE and Coomassie Brilliant Blue staining.
Cryo-EM sample preparation and data acquisition For cryo-EM grid preparation, 3.0 μL of purified DiCas7-11-crRNA, DiCas7-11 Inactive -crRNA, and DiCas7-11-crRNA-TPR-CHAT complexes were applied to glow-discharged (45 s) holey carbon grids (Quantifoil Au R2/1, 200 mesh).The grids were blotted for 3.0 s and then frozen by plunging into liquid ethane that had been cooled with liquid nitrogen using a Vitrobot Mark IV system (Thermo Fisher Scientific) at 4 °C and 100% humidity.The grids were loaded onto a Titan Krios cryogenic electron microscope (Thermo Fisher Scientific) operating at 300 kV.The microscope equipped with a 50 μm condenser lens aperture was set to a spot size of 6 and magnification at 165,000× (corresponding to a calibrated sample at 0.85 Å per physical pixel) and a K2 direct electron device equipped with a BioQuantum energy filter operated at 20 eV (Gatan).Movie stacks were collected automatically using EPU2 software (Thermo Fisher Scientific) with the K2 detector operating in counting mode.The recording rate was set to 5 raw frames per second, and the total exposure time was set to 6 s, resulting in 30 frames per stack and a total dose of ~60 e-/Å2.A total of 3232 movie stacks were collected for the DiCas7-11-crRNA-TPR-CHAT complex, 2997 movie stacks were collected for DiCas7-11-crRNA, and 1913 movie stacks were collected for DiCas7-11 Inactive -crRNA.The defocus range during data collection spanned from −1.0 to −1.8 μm.

Image processing and 3D image reconstruction
Cryo-EM data processing was conducted using EMAN (v2.31), 37 Relion (v4.0) and cryoSPARC (v4.1). 38,39For the DiCas7-11-crRNA-TPR-CHAT complex, a total of 3232 movie stacks were collected.All 30 frames in each stack were added with motion correction using MotionCorr2 40 and double binning to attain a pixel size of 0.85 Å per pixel.Dose weighting was also applied.Visual inspection of each micrograph was conducted to remove poorquality images due to contamination from crystalline ice or other visible contaminants.After contrast transfer function (CTF) correction using CTFFIND4, 41 3228 micrographs were processed for neural network particle picking in EMAN2.2.A total of 622,076 particles were isolated in Relion4 using a 320 pixel box.The extracted particles were downscaled twice and subjected to reference-free 2D classification to eliminate false picks and obvious junk classes.After two rounds of 2D classification, approximately 499,545 particles in the best classes were visually examined and selected for further analysis.These particles were then input into cryoSPARC to generate an initial model.All particles were divided into six classes, and two classes showed distinct protein profiles after ab initio reconstruction and heterogeneous refinement in cryoSPARC.One class contained 78,984 particles (15.8% of the total), and the other class contained 147,448 particles (29.5% of the total).These two classes were imported into Relion for autorefinement and Bayesian polishing.Finally, 3D nonuniform refinement and local refinement options in cryoSPARC were employed to determine the global resolution of the DiCas7-11-crRNA-TPR-CHAT map (2.90 Å) and DiCas7-11-crRNA-TPR-CHAT NTD map (2.86 Å).The resolutions for these final maps were estimated using the 0.143 criterion of the Fourier shell correlation (FSC) curve in cryoSPARC.The final maps were subjected to low-pass filtering and visualized in UCSF Chimera. 42or the DiCas7-11-crRNA complex, a total of 2997 movies were collected.From these movies, 529,643 particles were extracted using cryoSPARC and categorized into six classes for ab initio reconstruction and heterogeneous refinement.Of these, 216,156 particle (representing 40.8% of total) from the selected optimal class were processed through final homogeneous refinement, nonuniform refinement, and local refinement.The resulting density map exhibited a global resolution of 2.93 Å.
For the DiCas7-11 Inactive -crRNA complex, a total of 1913 movies were collected.CryoSPARC was used to extract 566,672 particles, which were then classified into five classes for ab initio reconstruction and heterogeneous refinement.Among these, 106,871 particle projections (50.1% of the total) from the optimal class were subjected to further processing, including focused 3D classification in Relion.Ultimately, 21,836 particles were selected for nonuniform refinement and local refinement in cryoSPARC.The resulting density map showed a global resolution of 3.53 Å.

Fig. 1
Fig. 1 DiTPR-CHAT inhibits the cleavage activity of DiCas7-11.a A domain structure illustration of the DiType III-E CRISPR-Cas system.b Diagram illustrating target RNA cleavage by DiCas7-11-crRNA or inhibition by DiTPR-CHAT.c In vitro analysis of target RNA cleavage by DiCas7-11 at varying molar concentrations of (1×, 2× or 4×) in the presence of crRNA with or without DiTPR-CHAT (1×, 1.5× or 3×) (n = 3).The DiCas7-11-crRNA was preincubated with DiTPR-CHAT before adding the target RNA.The control consists of the labeled target RNA only

Fig. 5
Fig. 5 The mechanism by which DiTPR-CHAT NTD inhibits the activity of DiCas7-11.a EMSA of target DiCas7-11 Inactive with DiTPR-CHAT (Full length, F), DiTPR-CHAT NTD (NTD), and DiTPR-CHAT CTD (CTD) in the presence of crRNA and labeled target RNA (n = 3).The control sample consisted of DiCas7-11 Inactive , crRNA and target RNA.The bands indicated by an asterisk (*) is a non-specific band that appear constantly in EMSA, which may be from the degradation of target RNA.b In vitro target RNA cleavage assay by crRNA-DiCas7-11 (at 2×, 4× or 8× molar concentrations) preincubated with DiTPR-CHAT NTD (at 0.5×, 2× or 8× molar concentrations).The control sample consisted of only labeled target RNA.c Model of DiCas7-11 showing inhibition by DiTPR-CHAT NTD .In the absence of DiTPR-CHAT NTD , DiCas7-11 adopts a relaxed conformation and exhibits optimal cleavage activity.When bound to DiTPR-CHAT NTD , DiCas7-11 presents a compact conformation (Cas7.4(V1371-R1336) is visible), blocking target RNA from binding and reducing cleavage activity